Bottom Line:
Elevated oxidative stress in cancer cells contributes to hyperactive proliferation and enhanced survival, which can be exploited using agents that increase reactive oxygen species (ROS) beyond a threshold level.NAC pre-treatment reversed inhibition of mTORC1 targets, demonstrating a ROS-dependent mechanism.Overall, our results illustrate the importance of disruption of the intrinsically high oxidative stress in melanoma cells to selectively inhibit their survival mediated by PI3K/AKT/mTOR.

Mentions:
Recent work from our laboratory suggests that NexrutineR modulates ROS in pancreatic cancer cells [22]. As such, we evaluated the potential of NexrutineR to disrupt the oxidative stress threshold in melanoma cells. Using fluorescence microscopy we found that total ROS levels (carboxydichlorofluorescein) increased in a dose-dependent manner after treatment with NexrutineR (Figure 2A). Quantification of the imaging data showed an increase in the percentage of ROS-positive cells in all three melanoma cell lines (Suppl. Figure 2A). Validation of ROS levels using flow cytometry showed a higher percentage of ROS-positive cells after 10 and 20 μg/ml NexrutineR treatment for 3 h compared with control cells (data not shown). To evaluate the source of ROS from NexrutineR treatment, we determined mitochondrial superoxide production using the Mitosox red fluorescent indicator. We found that NexrutineR treatment (10, 20 μg/ml; 3 h) resulted in a significant increase in mitochondrial superoxide production in all the melanoma cells that appeared to saturate at 10 μg/ml NexrutineR (Figure 2B). Interestingly, we did not observe increased superoxide levels in the melanocyte line at 10 μg/ml NexrutineR, and a non-significant increase at 20 μg/ml (Figure 2B). Using PO-1 dye as a probe for H2O2 production by NexrutineR, we found induction of H2O2 in all melanoma cells, which is an increment over the basal levels (Figure 2C). Further, we examined the effect of NexrutineR on intracellular redox balance, using reduced and oxidized glutathione as markers. Strikingly, we found that NexrutineR treatment resulted in increased glutathione ratio (GSH:GSSG) in melanocytes, which is indicative of reduced glutathione (Figure 3A), which was similar as the effect of NAC treatment. However, NexrutineR treatment decreased glutathione ratios (GSH:GSSG) in all melanoma cells, indicating increased cellular oxidative stress (Figure 3B). NAC pre-treatment partially abrogated the NexrutineR-modulated glutathione ratio. Evaluation of the effect of NexrutineR on mitochondrial membrane uncoupling showed that in all melanoma cell lines, NexrutineR treatment (20 μg/ml; 3 h) resulted in significant decrease in mitochondrial membrane potentials (ΔΨ) compared with vehicle-treated cells (Figure 3C). Melanocytes on the other hand showed no significant effect on mitochondrial membrane potential (Figure 3C). Uncoupling of mitochondrial membranes following treatment are visible as reduced fluorescent intensity due to decreased sequestration of potentiometric dye TMRM in mitochondria, also determined by quantification of membrane potentials (Suppl. Figure 3A). Lastly, we evaluated how NexrutineR affected the master regulator of oxidative stress; PGC1α. Our results show that NexrutineR treatment (20 μg/ml; 18 h) decreased PGC1α protein level in all melanoma cell lines unlike the HEMn melanocytes (Figure 3D). Quantification of the protein level is shown in Supplementary Figure 3B. Taken together, these data reveal the differential redox response of melanocyte and melanoma cells to NexrutineR-mediated oxidative stress induction; wherein melanoma cells unlike melanocytes do not activate the antioxidant response leading to increased oxidative stress.

Mentions:
Recent work from our laboratory suggests that NexrutineR modulates ROS in pancreatic cancer cells [22]. As such, we evaluated the potential of NexrutineR to disrupt the oxidative stress threshold in melanoma cells. Using fluorescence microscopy we found that total ROS levels (carboxydichlorofluorescein) increased in a dose-dependent manner after treatment with NexrutineR (Figure 2A). Quantification of the imaging data showed an increase in the percentage of ROS-positive cells in all three melanoma cell lines (Suppl. Figure 2A). Validation of ROS levels using flow cytometry showed a higher percentage of ROS-positive cells after 10 and 20 μg/ml NexrutineR treatment for 3 h compared with control cells (data not shown). To evaluate the source of ROS from NexrutineR treatment, we determined mitochondrial superoxide production using the Mitosox red fluorescent indicator. We found that NexrutineR treatment (10, 20 μg/ml; 3 h) resulted in a significant increase in mitochondrial superoxide production in all the melanoma cells that appeared to saturate at 10 μg/ml NexrutineR (Figure 2B). Interestingly, we did not observe increased superoxide levels in the melanocyte line at 10 μg/ml NexrutineR, and a non-significant increase at 20 μg/ml (Figure 2B). Using PO-1 dye as a probe for H2O2 production by NexrutineR, we found induction of H2O2 in all melanoma cells, which is an increment over the basal levels (Figure 2C). Further, we examined the effect of NexrutineR on intracellular redox balance, using reduced and oxidized glutathione as markers. Strikingly, we found that NexrutineR treatment resulted in increased glutathione ratio (GSH:GSSG) in melanocytes, which is indicative of reduced glutathione (Figure 3A), which was similar as the effect of NAC treatment. However, NexrutineR treatment decreased glutathione ratios (GSH:GSSG) in all melanoma cells, indicating increased cellular oxidative stress (Figure 3B). NAC pre-treatment partially abrogated the NexrutineR-modulated glutathione ratio. Evaluation of the effect of NexrutineR on mitochondrial membrane uncoupling showed that in all melanoma cell lines, NexrutineR treatment (20 μg/ml; 3 h) resulted in significant decrease in mitochondrial membrane potentials (ΔΨ) compared with vehicle-treated cells (Figure 3C). Melanocytes on the other hand showed no significant effect on mitochondrial membrane potential (Figure 3C). Uncoupling of mitochondrial membranes following treatment are visible as reduced fluorescent intensity due to decreased sequestration of potentiometric dye TMRM in mitochondria, also determined by quantification of membrane potentials (Suppl. Figure 3A). Lastly, we evaluated how NexrutineR affected the master regulator of oxidative stress; PGC1α. Our results show that NexrutineR treatment (20 μg/ml; 18 h) decreased PGC1α protein level in all melanoma cell lines unlike the HEMn melanocytes (Figure 3D). Quantification of the protein level is shown in Supplementary Figure 3B. Taken together, these data reveal the differential redox response of melanocyte and melanoma cells to NexrutineR-mediated oxidative stress induction; wherein melanoma cells unlike melanocytes do not activate the antioxidant response leading to increased oxidative stress.

Bottom Line:
Elevated oxidative stress in cancer cells contributes to hyperactive proliferation and enhanced survival, which can be exploited using agents that increase reactive oxygen species (ROS) beyond a threshold level.NAC pre-treatment reversed inhibition of mTORC1 targets, demonstrating a ROS-dependent mechanism.Overall, our results illustrate the importance of disruption of the intrinsically high oxidative stress in melanoma cells to selectively inhibit their survival mediated by PI3K/AKT/mTOR.